- MA5-27879 - Invitrogen Antibodies XPO1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of CRM1 was performed by loading whole-cell lysates of four different dog OSA cell lines, five human OSA cell lines and non-transformed human osteoblasts (HFOB). CRM1 was detected at approximately 123 kDa using a CRM1 Monoclonal Antibody (Product # MA5-27879) at a dilution of 1:1000 in Blocking Buffer (Product # 37543) overnight at 4°C on a rocking platform, followed by a 1-hour room-temperature incubation of a goat anti-mouse IgG antibody at a dilution of 1:5000. GAPDH were stained as loading control. Data courtesy of Antibody Data Exchange Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of CRM1 in Lane 1: rat liver tissue lysate, Lane 2: rat lung tissue lysate, Lane 3: mouse liver tissue lysate, Lane 4: mouse lung tissue lysate, Lane 5: Rabbit IgG, Lane 6: Marker 1113, Lane 7: human HepG2 whole cell lysate, Lane 8: human SMMC-7721 whole cell lysate, Lane 9: human HeLa whole cell lysate, Lane 10: human JURKAT whole cell lysate using 50 µg (reducing conditions) per well. Electrophoresis was performed on 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours and protein was transferred to a nitrocellulose membrane at 150mA for 50-90 minutes. Sample was blocked with 5% Non-fat Milk/TBS for 1.5 hours at room temperature, incubated with CRM1 monoclonal antibody (Product # MA5-27879) at a dilution of 0.5 µg/mL (overnight at 4°C), followed by goat anti-mouse IgG-Biotin secondary antibody at a dilution of 1:10,000. Signal development was performed using a chemiluminescence (ECL) kit.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of CRM1 was performed by loading whole-cell lysates of four different dog OSA cell lines, five human OSA cell lines and non-transformed human osteoblasts (HFOB). CRM1 was detected at approximately 123 kDa using a CRM1 Monoclonal Antibody (Product # MA5-27879) at a dilution of 1:1000 in Blocking Buffer (Product # 37543) overnight at 4°C on a rocking platform, followed by a 1-hour room-temperature incubation of a goat anti-mouse IgG antibody at a dilution of 1:5000. GAPDH were stained as loading control. Data courtesy of Antibody Data Exchange Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of CRM1 in, Lane 1: rat liver tissue lysates, Lane 2: rat lung tissue lysates, Lane 3: mouse liver tissue lysates, Lane 4: mouse lung tissue lysates, Lane 5: human HepG2 whole cell lysates, Lane 6: human SMMC-7721 whole cell lysates, Lane 7: human Hela whole cell lysates, Lane 8: human Jurkat whole cell lysates. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 µg of sample under reducing conditions. After Electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. The membrane was blocked with 5% non-fat milk/TBS for 1. 5 hour at RT. The membrane was incubated with CRM1 Monoclonal Antibody (5G3) (Product # MA5-27879) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0. 1% Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1. 5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit. A specific band was detected for CRM1 at approximately 123 kDa. The expected band size for CRM1 is at 123 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of XPO1 was achieved by transfecting U-2 OS with XPO1 specific siRNAs (Silencer® select Product # s14937). Western blot analysis (Fig. a) was performed using whole cell extracts from the XPO1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with CRM1 Monoclonal Antibody (5G3) (Product # MA5-27879, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 0.25µg/ml, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to XPO1. An uncharacterized band (*) was observed at ~100 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using anti-CRM1 Monoclonal Antibody (5G3) (Product # MA5-27879) and a 125 kDa band corresponding to XPO1 was observed across cell lines and tissue extracts along with an uncharacterized band (*) at ~95 kDa in the tested samples. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), Jurkat (Lane 2), A549 (Lane 3), HepG2 (Lane 4), U2OS (Lane 5), tissue extracts (30ug lysate) of Mouse Ovary (Lane 6), Mouse Testis (Lane 7) and Mouse Heart (Lane 8) were electrophoresed using NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001).The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry analysis of CRM1 using anti-CRM1 antibody (Product # MA5-27879). CRM1 was detected in a section of U2OS cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum and then incubated with 2μg/mL mouse anti- CRM1 antibody (Product # MA5-27879) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of CRM1 on paraffin-embedded human intestinal cancer tissue. Antigen retrieval was performed using citrate buffer (pH6, epitope retrieval solution) for 20 mins. Sample was blocked using 10% goat serum, incubated with CRM1 polyclonal antibody (Product# MA5-27879) with a dilution of 2 µg/mL (overnight at 4°C), followed by biotinylated goat anti-mouse IgG (30 minutes at 37°C). Development was performed using Streptavidin-Biotin-Complex (SABC) with DAB chromogen method.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow Cytometry of CRM1 in SiHa cells (blue line), isotype control mouse IgG (green line), and unlabeled (red line). Samples were blocked with 10% goat serum, incubated with CRM1 Monoclonal Antibody (5G3) (Product # MA5-27879) at a dilution of 1 μg (per 1x10^6 cells), followed by DyLight®488 conjugated goat anti-rabbit IgG (for 30 minutes at 20°C) using 5-10 μg (per 1x10^6 cells) dilution.

MA5-27879